Infection‐induced seroconversion and seroprevalence of SARS‐CoV‐2 among a cohort of children and youth in Montreal, Canada

Abstract The EnCORE study is a prospective serology study of SARS‐CoV‐2 in a cohort of children from Montreal, Canada. Based on data from our fourth round of data collection (May–October 2022), we estimated SARS‐CoV‐2 seroprevalence and seroconversion. Using multivariable regression, we identified factors associated with seroconversion. Our results show that previously seronegative children were approximately 9–12 times more likely to seroconvert during the early Omicron‐dominant period compared to pre‐Omicron rounds. Unlike the pre‐Omicron rounds, the adjusted rate of seroconversion among 2‐ to 4‐year‐olds was higher than older age groups. As seen previously, higher seroconversion rates were associated with ethnic/racial minority status.


| INTRODUCTION
SARS-CoV-2 transmission dynamics shifted dramatically in 2022 with the dominance of highly-infectious Omicron variants and the loosening of preventative measures such as masking and social distancing. 1 Disease surveillance systems had also changed, with PCR testing largely replaced by rapid antigenic testing of individuals along with population wastewater surveillance. 2 In this context, longitudinal serological data could indicate the magnitude of infection-induced SARS-CoV-2 seropositivity as well as determinants of seroconversion, which can help identify subpopulations that are vulnerable to infection. The EnCORE study is a prospective serology study of SARS-CoV-2 in children recruited from schools in four neighbourhoods in Montreal Canada. We present SARS-CoV-2 seroconversion and seroprevalence results from the fourth round of data collection (May to October 2022) and identify factors associated with the risk of seroconversion. Recruitment was re-opened in Rounds 2 and 4. Participating children provided a dried blood spot (DBS) sample for serology testing, and parents provided household data through online questionnaires. 3 Laboratory testing for seropositivity was based on enzyme-linked immunosorbent assays using the receptor-binding domain from the spike protein, the spike protein and the nucleocapsid protein, as antigens (Appendix S1).
In this analysis, we used Round 4 data, which included 726 participants aged 2 to 19 years old. Three hundred and ninety-six of these children had been found seronegative in Round 3. We provide seroprevalence estimates for all 726 children in Round 4 and seroconversion rates between Rounds 3 and 4, for the 396 children that contributed to both rounds. Seroconversion was defined as the number of children seronegative in Round 3 and seropositive in Round 4. The seroconversion rate was the number of newly seropositive children in Round 4 divided by the sum of participants' time at risk between the third and fourth round DBS dates, in years. If a participant had a positive PCR or rapid antigen test, the test date (rather than the fourth round DBS date) marked the end of the participant's at-risk time. To give context to the pandemic experience in Montreal and for our cohort of children through time, we present the Bank of Canada stringency index for Quebec, a percentage reflecting the strictness of public health measures, and for Rounds 2 to 4, the EnCORE cohort seroconversion rates and percentage of each age group that was vaccinated.
To assess the effect of each variable on the rate of seroconversion, we used multivariable quasi-Poisson regressions after imputing data for missing values. To mitigate effects of loss to follow up, we applied inverse probability of censoring weights (Appendix S1). Unadjusted seroprevalence estimates for Round 4 by age, sex, vaccination status and prior seropositivity status were also estimated with correction by inverse probability of censoring weights. For further details on the study design and laboratory testing, please refer to Zinszer et al. and Zinszer et al., [3][4][5] and for the statistical methods, see Appendix S1.

| RESULTS
The infection-induced seroconversion rate was 133 per 100 P-Y (N = 396) among returning children, seronegative in Round 3; approximately 9-12 times that of pre-Omicron rounds ( Figure 1). Relative to F I G U R E 1 EnCORE cohort seroconversion rate per 100 person-years (P-Y) (light grey), percentage of Encore sample vaccinated with at least one dose, by age group (blues) and the Bank of Canada stringency index (%) for Quebec (dark grey) through Rounds 2, 3 and 4, with Omicron dominance starting in mid-December 2021. the oldest age group, a 36% higher seroconversion rate was observed in 2-to 4-year-old children and a 24% lower rate in 5-to 11-year-old children (Table 1). This could largely be explained by age group differences in vaccination uptake. Vaccination was open to 12-19-year-olds in June 2021 and 98% were vaccinated prior to Round 4; 5-11-year-olds were eligible from November 2021 and 91% were vaccinated by Round 4, and the 2-to 4-year-olds could be vaccinated as of July 2022 and 2% were vaccinated by Round 4 ( Figure 1). All vaccinated 2-4-year-olds had only one dose, over 83% of 5-11-year olds had two or more doses, and over 86% of the 12-19-year olds had two or more doses. Moreover, the percentage of the age groups having a vaccination within 120 days of their DBS was 2% for 2-4-year-olds, 32% for 5-11-year-olds and 27% for 12-19-year-olds, suggesting greater protection for the older participants. All 19 children that were previously seropositive in either Round 1 or 2 (but seronegative in Round 3), were found to be seropositive again in Round T A B L E 1 Crude seroconversion rates and adjusted seroconversion rate ratios, corrected for loss to follow-up, for study population characteristics, estimated by multivariable quasi-Poisson regression, for the fourth round of data collection (May to October 2022) of the EnCORE study.  Differences in seroconversion rates were observed by age group.
Consistent with other studies, we found that young children were more affected by Omicron's surge with an estimated 36% higher rate of seroconverting, which was not seen in pre-Omicron rounds. [4][5][6] This may be explained by the high vaccine uptake in the older age groups.
In the seroconversion cohort, only 2% of 2-4-year-olds were vaccinated and with only one dose. Older participants had a vaccine uptake exceeding 90%. Though older children may have been vaccinated over a year before their fourth round DBS, and BNT162b2 vaccine effectiveness against Omicron may wane significantly by 120 days, 12 approximately one third of the older children had a second, third or fourth dose within 120 days of their DBS. This finding was consistent with CDC data demonstrating greater increases in seroprevalence between September and February 2022 among age groups with the lowest vaccination rates. 13 We were not able to look at vaccination status in greater depth for our seroconversion analysis, given the multicollinearity with age groups, although this certainly warrants further exploration.
We found that having a parent self-identify as a racial or ethnic minority was associated with a 44% higher rate of seroconverting during this period than children with white parents. This was also an important risk factor for seroconversion in EnCORE's earlier rounds and has been highlighted on multiple occasions since the beginning of the pandemic as a major risk factor of infection and infection severity. 4,5,14 Our results support that even in the context of Omicronenhanced contagiousness, health inequalities still play a key role in SARS-CoV-2 incidence.
Two thirds of participants who were previously seropositive in any round were also seropositive in Round 4. All 19 children that were seropositive in the first or second round but seronegative in the third were again seropositive in Round 4. This could indicate a reinfection or the persistence of a detectable antibody response from an earlier infection. We previously found that the likelihood of remaining seropositive was 68% at 6 months versus 41% at 1 year; estimates vary in other studies of children and adults, with as many as 96% of children and 83% of adults having detectable antibodies at 11-12 months after infection. 5,15 A limitation of our study was the significant loss to follow-up and based on anecdotal reports from parents, this was largely associated with the parent-child discomfort in providing the DBS. However, we corrected estimates using inverse probability of censoring weights and we found little evidence of differential patterns in loss to followup. 5 Nevertheless, the analysis was not powered to detect meaningful differences in seroconversion rates. Kate Zinszer had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

ACKNOWLEDGEMENTS
We sincerely thank all of the children, youth and their parents for their precious contribution to our study. We also thank all of the day cares, schools and school boards for their help with recruitment. We are grateful for the support and guidance of our partners: Direction régionale de santé publique de Montréal, COVID-19 Immunity Task Force, Institut national de santé publique du Québec and Observatoire pour l'éducation et la santé des enfants.

CONFLICT OF INTEREST STATEMENT
JP reports grants from MedImmune, grants and personal fees from Merck and AbbVie, and personal fees from AstraZeneca, all outside the submitted work.

DATE AVAILABILITY STATEMENT
Data are available on reasonable request from the corresponding author.

ETHICAL APPROVAL STATEMENT
The research ethics boards of the Université de Montréal and the Centre Hospitalier Universitaire Sainte-Justine approved this study in accordance with the laws of the Government of Quebec. All parents and children gave consent prior to participation.